Expression and activation of nuclear hormone receptors result in neuronal differentiation and favorable prognosis in neuroblastoma

Background Neuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR). Methods Cell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lipid droplets by Nile Red staining. Expression of angiogenesis, proliferation, and neuronal differentiation markers, and tumor sections were assessed by immunohistochemistry. Gene expression from NB patient as well as adrenal gland cohorts were analyzed using GraphPad Prism software (v.8) and GSEA (v4.0.3), while pseudo-time progression on post-natal adrenal gland cells from single-nuclei transcriptome data was computed using scVelo. Results Here, we show that simultaneous activation of GR and ERα potentiated induction of neuronal differentiation, reduced NB cell viability in vitro, and decreased tumor burden in vivo. This was accompanied by a metabolic reprogramming manifested by changes in the glycolytic and mitochondrial functions and in lipid droplet accumulation. Activation of the retinoic acid receptor alpha (RARα) with all-trans retinoic acid (ATRA) further enhanced the differentiated phenotype as well as the metabolic switch. Single-cell nuclei transcriptome analysis of human adrenal glands indicated a sequential expression of ERα, GR, and RARα during development from progenitor to differentiated chromaffin cells. Further, in silico analysis revealed that patients with higher combined expression of GR, ERα, and RARα mRNA levels had elevated expression of neuronal differentiation markers and a favorable outcome. Conclusion Together, our findings suggest that combination therapy involving activation of several NHRs could be a promising pharmacological approach for differentiation treatment of NB patients. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-022-02399-x.


C) Quantification of neurites in the experiment shown in B.
Data is presented as number of neurites per cell in three different images from three independent experiments. D) Western blot of the indicated proteins in BE(2)-EV and BE(2)-GR cells following treatment with 100 nM DEX, 0.5 μM ATRA, or their combination during seven days. Proteins were separated on two gels with β-actin as loading control. Molecular weight markers shown to the left. Representative blots from three independent experiments. E) Densiometric analysis of Western blots from three independent experiments of the indicated proteins from BE(2)-EV and BE(2)-GR represented in D (MYCN) and Figure  1B. F) mRNA expression levels of the indicated genes in BE(2)-EV and BE(2)-GR cells following treatment with 100 nM DEX, 0.5 μM ATRA, or their combination during seven days. Β2-microglobulin (B2M) and β-actin were used as control genes. Data in A and C, E, and F are presented as mean ± SD of at least three independent experiments; statistical analysis: t-test, with *, **, ***, and **** indicating p < 0.05, p < 0.01, p < 0.001, and p < 0.0001. loading control. Molecular weight markers shown to the left. Representative blots from three independent experiments. B) Densiometric analysis of c-MYC protein from parental SH-SY5Y and SH-SY5Y-GR as well as from parental SK-N-AS and SK-N-AS-GR from the Western blots represented in A. C) Neurite outgrowth assay after seven days treatment with 100 nM DEX, 5 μM ATRA, or their combination. Representative phase contrast microscopy images from three independent experiments. Scale bars indicate 20 µm. D) Densiometric analysis of the indicated proteins from parental SH-SY5Y and SH-SY5Y-GR from the Western blots represented in Figure 1E. E) Densiometric analysis of the indicated protein from parental SK-N-AS and SK-N-AS-GR from the Western blots represented in Figure 1G.
The densitometric analyses in B, D, and E were performed from Western blots from three independent experiments and are shown as mean ± SD; statistical analysis: t-test, with *, **, indicating p < 0.05 and p < 0.01, respectively.

Supplementary Figure 3 related to Figure 2.
Tumor volume for each mouse in the xenograft experiment comparing tumors from BE(2)-EV (blue) and BE(2)-GR (red) cells, respectively; n= 6 in the BE(2)-EV group and n= 7 in the BE(2)-GR group. Tumor growth was followed until the control group reached the ethical endpoint volume of 1 cm 3 . The summary of the experiment is shown in Figure 2A. B) Viability graphs for IC50 calculation of DEX, E2, and ATRA after 72 h of treatment with the indicated concentrations in BE(2)-GR+EV (blue) and BE(2)-GR+ERα (red) cells. C) Percentage of viable cells determined by WST-1 after activation of GR and ER⍺ in BE(2)-GR+EV (blue) and BE(2)-GR+ERα (green) cells. Viability is presented as 100 % compared to control levels. Cells were treated with E2, DEX, ATRA, or in combination as indicated for three days.

S O A T R A E t h a n o l D E X E 2 + D E X + A T R A E 2 + D E X E 2
Protein levels (relative to

BE(2)-GR+ER
controls. Statistical analysis: t-test, with * and *** indicating p < 0.05, and p < 0.001, respectively. C) Western blot of the indicated proteins in BE(2)-GR+EV and BE(2)-GR+ERα cells following treatment with 10 nM E2, 100 nM DEX, 0.5 μM ATRA, or their combination during seven days. Proteins were separated on two gels with β-actin as loading control. Molecular weight markers shown to the left. Representative blots from three independent experiments. D) Densiometric analysis of to Western blots from three independent experiments of the indicated proteins from BE(2)-GR+EV and BE(2)-GR+ERα presented in C (MYCN) and in Figure 3B. Data is shown as mean ± SD of two (p75 NTR ) or three independent experiments; statistical analysis: t-test, with *, **, and **** indicating p < 0.05, p < 0.01, and p < 0.0001.    Figure 4B. Data is shown as mean ± SD of three independent experiments; statistical analysis: t-test, with *, **, and *** indicating p < 0.05, p < 0.01, and p < 0.001.

S O A T R A E t h a n o l D E X E 2 + D E X + A T R A E 2 + D E X E 2 E 2
Protein levels (relative to -actin) Protein levels (relative to -actin)

S O A T R A E t h a n o l D E X E 2 + D E X + A T R A E 2 + D E X E 2 E 2
B) Densiometric analysis corresponding to Western blots from three independent experiments of the indicated proteins from IMR32-GR+EV and IMR32-GR+ERα cells represented in A (MYCN) and in Figure 4B. Data is shown as mean ± SD of three independent experiments; statistical analysis: t-test, with *, and **, indicating p < 0.05, p < 0.01. , providing a method to analyze key glycolytic parameters: glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. Glycolysis is defined by the ECAR rate reached after adding glucose. The maximum ECAR rate values after adding the ATP synthase inhibitor, Oligomycin, defines the glycolytic capacity. The glycolytic reserve is the ability to respond to an energy demand and it is the difference between the glycolytic reserve and glycolysis. By adding 2-deoxy-

ECAR (mpH/min/ g protein)
glucose (2-DG), it is confirmed that ECAR produced in the experiment is due to glycolysis. The non-glycolytic acidification is the ECAR produced by other sources than glycolysis. B) Graphical presentation of the principle of Mito stress test. This assy is used to assess the mitochondrial function by measuring the oxygen consumption rate (OCR), analyzing several mitochondrial parameters: basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. The initial OCR value represent basal cellular respiration. The decrease in OCR after injecting oligomycin correlates to the basal respiration used to ATP production. Proton leak is calculated by the remaining basal respiration not linked to ATP production and that could indicate mitochondrial damage. The maximal OCR reached after injecting the uncoupler agent carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and that produces the collapse of the proton gradient represents the maximal respiration. The spared respiratory capacity is the difference between maximal and basal respiration, indicating the flexibility of a cell to adapt to energy demand. The OCR value after addition of rotenone and antimycin A is considered as non-mitochondrial respiration.

C-D) Baseline and stressed metabolic phenotypes (indicated as open and filled squares, respectively) of C) BE(2)-GR+EV and D) BE(2)-GR+ERα cells after treatment during 72
h with ethanol, 10 nM E2, 100 nM DEX, or the combination of 10 nM E2 + 100 nM DEX. Quantification is presented in Figure 5E and Figure 5G.

E-F) Baseline and stressed metabolic phenotype (indicated as open and filled squares, respectively) of E) BE(2)-GR+EV and F) BE(2)-GR+ERα cells after treatment during 72
h with DMSO, ethanol, 0.5 μM ATRA, or the triple combination of 10 nM E2 + 100 nM DEX + 0.5 μM ATRA. Quantification is presented in Figure 5F and Figure 5H. All experiments in C-F were carried out three independent times. Images in A-B are adapted with permission from Seahorse Bioscience, Agilent Technologies, North Billerica, MA, USA. t-test with ns, **, and ***, indicating non-significant, p < 0.05, p < 0.01, p < 0.001, respectively. C) Gene set enrichment plots of differentiation and metabolic related processes comparing genes expressed in patients with combined high versus low GR and ERα mRNA expression levels (High GR+ERa versus Low GR+ERa ) from the SEQC cohort. Plots were obtained from GSEA (v4.0.3) using the C2 curate set collection presented in Additional File 1. FDR q-